The search for a vaccine against AIDS remains a major issue since the discovery of the HIV-1 (Barre-Sinoussi et al., 1983). The failure of classical vaccine approaches targeting the HIV-1 envelop proteins points out the interest to use another target such as the HIV-1 trans-activator of transcription protein called Tat, due to its extra cellular function involving the collapse of the immune cellular response against HIV infected cells (Jeang et al., 1999).
Tat exists predominantly in two different lengths, 86-87 residues or 99-101 residues, that display multifaceted activities (Jeang et al., 1999). The long forms are predominant in clinical isolates from all HIV-1 subtypes except subtype D, due to the presence of a non-synonymous single nucleotide polymorphism, creating a stop codon in the second exon encoding sequence (Jeang et al., 1999). Tat is divided into six regions (Kuppuswamy et al., 1989) with one called basic region being involved in most of the Tat activities. NMR studies of biologically active Tat variants revealed that the basic region and the other functional regions are well exposed to solvent and surround a core composed of part of the N-terminus with the well conserved Trp 11 (Péloponèse et al., 2000; Gregoire et al., 2001, Watkins et al., 2008). Among different Tat variants this folding is similar in aqueous solution but can change dramatically when exposed to hydrophobic solvent (Péloponèse et al., 1999). Tat is a flexible protein and structural changes are necessary for it to bind to its pharmacological targets (Loret et al., 1992).
Tat is found mainly in the nucleus of infected cells where it functions as a trans-acting transcriptional activator (Wong-Staal et al, 1985; Fujisawa et al., 1985), where it is known to be involved in the initiation of transcription and RNA chain elongation (Cullen, 1990) by a complex process involving interactions with cellular proteins and a stem-bulge loop leader RNA, TAR (trans-activation responsive region), on the viral mRNA and acetylation of the Tat (Bres et al., 2002). Studies have shown that it also participates with the reverse transcription of HIV-1 RNA (Harrich et al., 1997).
Despite the lack of a signal sequence, Tat is the only HIV-1 protein to be secreted by HIV-1-infected cells and is found in detectable levels in the culture supernatants of HIV-1-infected cells (0.1-1 ng/ml) (Ensoli et al., 1990; Westendorp et al., 1995b; Chang et al, 1997) and in the sera of HIV-1-infected patients (1-40 ng/ml) (Xiao et al., 2000; Westendorp et al., 1995b). Extracellular Tat display multifaceted activities (Jeang et al., 1999), but the most important is to trigger apoptosis of uninfected T cells by traversing the cell membrane, leading to apoptosis through the mitochondrial pathway (Chen et al., 2002, Campbell et al., 2004, de Mareuil et al., 2005). Neutralization of extracellular Tat in vivo in SHIV-1 challenged macaques induces a rise of CD8+ T cells and HIV-1 infected CD4+ T cells become undetectable (Watkins et al., 2006).
Thus, the role of Tat in HIV-1 pathogenesis is not only as an essential protein for HIV-1 replication in infected cells, but also as an extracellular toxin (Gallo, 1999). Therefore, it is relevant to develop a vaccine targeting Tat (Goldstein, 1996). However, seropositive patients that have antibodies against Tat are unable to recognize Tat variants from all HIV-1 subtypes (Campbell et al., 2007b). Moreover, these antibodies fail to slow disease progression to AIDS (Senkaali et al., 2008).
It is clear that a Tat vaccine using a B subtype Tat found mainly in Europe and North America as previously proposed (Godstein, 1996, Zagury et al., 1998, Cafaro et al., 1999) has a low probability to provide a therapeutic and a preventive effect against HIV-1 infection in the rest of the world and particularly in Africa. Moreover, a Tat vaccine using a B subtype Tat has also a low probability to provide a therapeutic and a preventive effect even in Europe and North America due to the incapacity of the immune system to neutralize extra cellular Tat.
The two main vaccine strategies against Tat up to now use a short, 86 residue version of a B-subtype European Tat variant that is either inactivated (Zagury et al., 1998) or has full activity (Cafaro et al., 1999). These two approaches were tested on macaques followed by a homologous SHIV-1 challenge (Cafaro et al., 1999; Pauza et al., 2000). A significant decrease of viremia was observed in these two studies carried out respectively on Cynomolgus (Cafaro et al., 1999) and Rhesus macaques (Pauza et al., 2000), without showing complete protection during primo-infection. Another study showed a long term control of infection following SHIV-1 challenge on Tat vaccinated Cynomolgus macaques (Maggiorella et al., 2004). These studies point out that vaccines using biologically active Tat appears to be a safe approach as indicated by safety studies carried out on monkeys in which no local or systemic toxicity or adverse effects were observed (Cafaro et al., 1999, 2001; Caselli et al., 1999, Goldstein et al., 2000).
It is interesting to note that conflicting results appears in Tat vaccine studies on macaques since no protection was observed with a SIV challenge (Allen et al., 2002) or a vaccination with a recombinant virus coding for a Tat-Rev protein (Verrier et al., 2002). These conflicting results could be explained by a different immunization regimens, viral stock, routes of viral challenge and animal species. The difference between SIV Tat and HIV-1 Tat in the first study and the probability that a Tat-Rev recombinant protein does not have the native Tat folding or the native Rev folding for the second study may explain the absence of protection. More puzzling, however, is the result of two other studies using similar viral vectors expressing Tat, Env and Gag that give opposite conclusions. One study shows the efficacy of vectored Tat, but not Gag and Env (Stittelaar et al., 2002), while another study showed efficacy of vectored Gag and Env, but not Tat (Liang et al., 2005). The main difference in the two studies is that one is using a homologous challenge using the Tat Bru sequence in both the vaccine and in the SHIV (Stittelaar et al., 2002) and the other a heterologous challenge with the Tat Bru sequence in the vaccine and Tat Jr in the SHIV (Liang et al., 2005). HIV-1 Jr and HIV-1 Bru are B subtypes (FIG. 1) but their Tat sequences have non-conservative mutations inducing conformational changes (Gregoire & Loret., 1996). Theses mutations between the vaccine and the virus use for the challenge might explain the lack of efficacy of the Tat vectored vaccine in the second study (Liang et al., 2005). It is clear that the second study more closely resembled reality since a vaccinated person will not likely be exposed to a homologous virus infection, but in that case why have homologous Gag and Env been used (Liang et al., 2005)?
Over the last 20 years, HIV-1 vaccine studies that target the HIV-1 envelope proteins have been tested using a homologous SHIV/macaque model and have met with success (Feinberg et al., 2002). However, clinical trials were not successful (Vanichseni et al., 2004). This is due to the high genetic diversity of HIV-1 and this is why heterologous SHIV challenge in macaques, with a genetically distinct virus, should be used to determine if a vaccine can be effective against HIV-1 infection in humans (Feinberg et al., 2002). If a successful homologous SHIV challenge is helpful to show the interest of Tat vaccination in vivo, the development of a Tat vaccine in humans worldwide has to take in account the genetic diversity of Tat. It is important to note that immunization with the B subtype Tat Bru does not stimulate an efficient response against Tat variants from A and C HIV-1 subtypes that corresponds to 75% of the contamination in the world (Opi et al., 2002).
The interest to develop a Tat vaccine rose with the discovery that seropositive long-term non-Progressor (LTNP) patients had a higher level of Tat antibodies than seropositive Rapid Progressor (RP) patients (Re et al., 1995; van Baalen et al., 1997; Zagurry et al., 1998, Re et al., 2001; Addo et al., 2001, Belliard et al., 2003). It is interesting to note that with a serum dilution of 1:1000, Tat Bru is recognized by only 30% of the RP patients in Europe (Zagury et al., 1998) and only 10 to 14% in Africa (Butto et al., 2003). This percentage can reach up to 50% in Africa if other Tat variants from subtypes A, C and D are tested (Campbell et al., 2007). This result outlines again how mutations in Tat variants can affect immunogenicity but it shows also that a large amount of seropositive patients are unable to recognize Tat. Furthermore Tat antibodies in African RP patients have no effect on the progression to AIDS (Sankali et al., 2008). It shows that, for a majority of HIV-1 infected patients, Tat is not recognized while this protein is present in their blood and patients who recognize Tat cannot neutralize this protein. The reason why Tat is so badly recognized by the human immune system could be due to sequence similarity of the basic region of Tat with epitopes found in human proteins such as the protamine. It is clear that triggering an immune response against the Tat basic region (region IV) would lead to autoimmune response on humans. Region IV is well conserved among Tat variants (FIG. 1) but it is not recognized by sera from HIV-1 infected patients (Campbell et al., 2007). It is interesting to note that two third of new born children from HIV-1 infected mother succeed to escape to HIV-1 infection (Beattie et al., 2001). They are seropositive up to 18 months old and then retro seroconvert. This high proportion of new-born infants that resist to HIV infection excludes genetic factors that could be due to an innate immunity against HIV. It could be possible that a repression of the immune system to recognize Tat may exist in adults but not among new-born infants since protamine appears with sexual maturation.
A better attention should be taken to natural immunity occurring in adults against HIV-1. Natural immunity against HIV-1 is observed in a low proportion of the human population and cover different mechanisms going from chemokine mutations to the capacity to produce neutralizing antibodies against HIV-1 envelope (see Marmor et al., 2006 for a review). Natural immunity can be innate or acquired, the latter being of course the most interesting for a vaccine development. Patients with natural immunity against HIV-1 can be exposed and persistently seronegative (EPS) or can be seropositive and long term non progressor (LTNP). In most cases this natural immunity turns out to be an innate immunity. However there is a very rare category of patients highly exposed to the virus that are resistant to HIV-1 due apparently to an acquired immunity. Kenyan sex workers who are EPS had been intensely studied and their resistance to HIV-1 appears to be related to their capacity to develop an efficient CD8 T cell response against HIV-1 (McMichael & Rowland-Jones, 2001). However, the paradox is that the CD8 T cell response in EPS Kenyan sew workers is five time lower in magnitude than that of seropositive Kenyan sex workers who ultimately develop AIDS (Alimonti et al., 2006). To make things even more puzzling, studies of similar cohorts of EPS patients in Ivory Cost, Vietnam and Cambodia show that they have no HIV-1 specific CD8 T cell response but a natural killer (NK) cell responses (Scott-Algara et al., 2003; Jennes et al., 2006), antibodies against HIV-1 envelop proteins (Nguyen et al., 2006) or cellular factors affecting viral entry steps (Saez-Cirion et al., 2006). Although it was believed that these Kenyan EPS patients had an innate immunity, it turned out that it was an acquired immunity at least for some of them who became seropositive after a lapse in sex work (Kaul et al., 2001).
More interesting was the study of an EPS cohort identified in Gabon (Huet et al., 1989). During the eighties in Africa, it was observed in a remote area of Gabon called “Haut Ogooué” that HIV-1 infected patients were not developing AIDS (Delaporte et al., 1988). An epidemiological survey was decided and carried out on 750 pregnant women for two years and 25 were identified as seropositive (Huet et al., 1989). From these 25 seropositive women, 23 retro seroconverted and became EPS during the two years of the survey. Although EPS patients have normally no detectable virus, it was possible to isolate and clone a HIV-1 strain from one patient called Oyi when she was still seropositive (Huet et al., 1989). Contrary to other EPS cohort of sex workers or drug users that were constituted many years after the first exposure to HIV, the Gabon cohort was constituted during the primo infection and explains why it was possible to clone a virus. All women infected with HIV-1 Oyi did retro seroconvert but maintained a CTL response against HIV-1 and had antibodies against P24 (Huet et al., 1989). The high proportion of EPS phenotype in this cohort (92%) indicates that the retro seroconversion was probably due to an acquired immunity and not an innate immunity. Ten years after the publication of this epidemiological survey, the 23 women were in good health and the HIV-1 or a sign of infection was no longer detectable in their blood. Furthermore HIV-1 infection appears to be very low in Gabon compared to other central African countries (Delaporte et al., 1996). HIV-1 Oyi has genes similar to regular HIV-1 strains except the tat gene, which had mutations never found in other Tat variants (Gregoire & Loret, 1996). Immunization with Tat Oyi raises antibodies in rabbits that are able to recognize different Tat variants even with mutations of up to 38%, which is not possible with other Tat variants (Opi et al., 2002). Tat Oyi appears to induce a humoral immune response against a three-dimensional epitope that are conserved in Tat variants and this humoral response could make possible to neutralize extracellular Tat. The role of extracellular Tat was not known during the eighties and the presence of antibodies against Tat was not tested in this Gabon cohort (Huet et al., 1989).
Seven rhesus macaques were immunized with synthetic Tat Oyi complemented with an adjuvant then a heterologous challenge with the European SHIV BX08 was carried out on Tat Oyi vaccinated macaques and control macaques. Tat Oyi vaccinated macaques had a lower viremia associated with a rise of the CD8 T cells compared with control macaques. Moreover, SHIV infected cells were no longer detectable at 8 weeks post-challenge in Tat Oyi vaccinated macaques. It is interesting to note that the macaque that had the lowest viremia had no antibodies against SHIV envelop proteins. The macaque was challenged again, made a short period of seropositivity and seroconverted (Watkins et al., 2006). It was therefore possible to reproduce experimentally on macaque what is observed in the field with EPS patients. It is important to note that this “EPS macaque” had a detectable viremia (watkins et al., 2006). This is a very promising result because it was a heterologous SHIV challenge and it shows that it is possible to dramatically reduce the level of HIV infected cells. This goal has never been achieved in seropositive patients under HAART treatment.